Toll-like receptor 4 initiates an innate immune response to lipopolysaccharide in human conjunctival epithelial cells.
Conjunctival epithelial cells serve as a first line of defense against pathogens presented to the innate immune system. The inflammatory response to Gram-negative bacteria is initiated by toll-like receptor 4 (TLR4). This study investigated whether a TLR4 ligand induces production of inflammatory cytokines in human conjunctival epithelial cells (HCECs) through nuclear factor kappa-B (NF-kappaB). HCECs were evaluated for TLR4 expression by reverse transcriptase-polymerase chain reaction, Western blot analysis, and flow cytometric analysis. HCECs were stimulated with various concentrations of lipopolysaccharide (LPS) and the innate immune response was quantified by measuring expression of the inflammatory cytokines IL-6 and IL-8. Functional NF-kappaB activation was examined using a luciferase reporter assay. Expression of TLR4-specific mRNA as well as its corresponding protein was observed in HCECs. Surface and intracellular expression of TLR4 was observed in flow cytometric analysis. Incubation of HCECs with LPS led to secretion of IL-6 and IL-8. Blockade of TLR4- and TNFR-associated factor (TRAF) 6 activity abolished LPS-induced inflammatory response in HCECs and incubation of HCECs with LPS led to activation of the NF-kappaB transcription factor. LPS did not enhance the TLR4 expression at both mRNA and protein levels in HCECs. Our results demonstrate that surface expression of TLR4 in HCECs can elicit a TLR4-mediated innate immune response through TRAF6-NF-kappaB and contribute to an inflammatory environment on the ocular surface.
Chung
So-Hyang
SH
Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
Kweon
Mi-Na
MN
Lee
Hyung Keun
HK
Choi
Seung-Il
SI
Yang
Jin-Young
JY
Kim
Eung Kweon
EK
D002478:Cells, Cultured; D003228:Conjunctiva / Q000276:immunology*; D016207:Cytokines / Q000096:biosynthesis; D004306:Dose-Response Relationship, Immunologic; D004847:Epithelial Cells / Q000276:immunology; D005136:Eye Proteins / Q000235:genetics / Q000276:immunology*; D015870:Gene Expression / Q000276:immunology; D006801:Humans; D007113:Immunity, Innate; D018928:Immunity, Mucosal; D007249:Inflammation / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis / Q000235:genetics; D008070:Lipopolysaccharides / Q000276:immunology; D016328:NF-kappa B / Q000276:immunology; D009363:Neoplasm Proteins / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology*
D016207:Cytokines; D005136:Eye Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009363:Neoplasm Proteins; D012333:RNA, Messenger; C120635:TAX1BP1 protein, human; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4